1 / 36

Protein Methods II

Protein Methods II. Andy Howard Introductory Biochemistry Fall 2009, IIT. Proteins are worth studying. We’ll finish our quick overview of methods of studying proteins. Plans. Purification methods Analytical methods Structural methods. Ion-exchange chromatography.

miya
Download Presentation

Protein Methods II

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript


  1. Protein Methods II Andy Howard Introductory BiochemistryFall 2009, IIT

  2. Proteins are worth studying • We’ll finish our quick overview of methods of studying proteins Biochemistry: Protein Methods II

  3. Plans • Purification methods • Analytical methods • Structural methods Biochemistry: Protein Methods II

  4. Ion-exchange chromatography • Charged species affixed to column • Phosphonates (-) retard (+)charged proteins:Cation exchange • Quaternary ammonium salts (+) retard (-)charged proteins: Anion exchange • Separations facilitated by adjusting pH Biochemistry: Protein Methods II

  5. Affinity chromatography • Stationary phase contains a species that has specific favorable interaction with the protein we want • DNA-binding protein specific to AGCATGCT: bind AGCATGCT to a column, and the protein we want will stick; every other protein falls through • Often used to purify antibodies by binding the antigen to the column Biochemistry: Protein Methods II

  6. Metal-ion affinity chromatography • Immobilize a metal ion, e.g. Ni, to the column material • Proteins with affinity to that metal will stick • Wash them off afterward with a ligand with even higher affinity • We can engineer proteins to contain the affinity tag:poly-histidine at N- or C-terminus Biochemistry: Protein Methods II

  7. High-performance liquid chromatography • Many LC separations can happen faster and more effectively under high pressure • Works for small molecules • Protein application is routine too, both for analysis and purification • FPLC is a trademark, but it’s used generically Biochemistry: Protein Methods II

  8. Electrophoresis • Separating analytes by charge by subjecting a mixture to a strong electric field • Gel electrophoresis: field applied to a semisolid matrix • Can be used for charge (directly) or size (indirectly) Biochemistry: Protein Methods II

  9. SDS-PAGE • Sodium dodecyl sulfate: strong detergent, applied to protein • Charged species binds quantitatively • Denatures protein • Good because initial shape irrelevant • Bad because it’s no longer folded • Larger proteins move slower because they get tangled in the matrix • 1/Velocity  √MW Biochemistry: Protein Methods II

  10. SDS PAGE illustrated Biochemistry: Protein Methods II

  11. Isoelectric focusing I • Protein applied to gel without charged denaturant • Electric field set up over a pH gradient (typically pH 2 to 12) • Protein will travel until it reaches the pH where charge =0 (isoelectric point) Biochemistry: Protein Methods II

  12. Isoelectric focusing II • Sensitive to single changes in charge (e.g. asp -> asn) • Can be readily used preparatively with samples that are already semi-pure Biochemistry: Protein Methods II

  13. Ultraviolet spectroscopy • Tyr, trp absorb and fluoresce:abs ~ 280-274 nm; f = 348 (trp), 303nm (tyr) • Reliable enough to use for estimating protein concentration via Beer’s law • UV absorption peaks for cofactors in various states are well-understood • More relevant for identification of moieties than for structure determination • Quenching of fluorescence sometimes provides structural information Biochemistry: Protein Methods II

  14. Warning: Specialty Content! • I determine protein structures (and develop methods for determining protein structures) as my own research focus • So it’s hard for me to avoid putting a lot of emphasis on this material • But today I’m allowed to do that, because it’s one of the stated topics of the day. Biochemistry: Protein Methods II

  15. How do we determine structure? • We can distinguish between methods that require little prior knowledge (crystallography, NMR, ?CryoEM?)and methods that answer specific questions (XAFS, fiber, …) • This distinction isn’t entirely clear-cut Biochemistry: Protein Methods II

  16. Crystallography: overview • Crystals are translationally ordered 3-D arrays of molecules • Conventional solids are usually crystals • Proteins have to be coerced into crystallizing • … but once they’re crystals, they behave like other crystals, mostly Biochemistry: Protein Methods II

  17. How are protein crystals unusual? • Aqueous interactions required for crystal integrity: they disintegrate if dried • Bigger unit cells (~10nm, not 1nm) • Small # of unit cells and static disorder means they don’t scatter terribly well • So using them to determine 3D structures is feasible but difficult Biochemistry: Protein Methods II

  18. Crystal structures: Fourier transforms of diffraction results • Experiment: • Grow crystal, expose it to X-ray • Record diffraction spots • Rotate through small angle and repeat ~180 times • Position of spots tells you size, shape of unit cell • Intensity tells you what the contents are • We’re using electromagnetic radiation, which behaves like a wave, exp(2ik•x) • Therefore intensity Ihkl = C*|Fhkl|2 Biochemistry: Protein Methods II

  19. What are these Fhkl values? • Fhkl is a complex coefficient in the Fourier transform of the electron density in the unit cell:(r) = (1/V) hklFhkl exp(-2ih•r) • Critical point: any single diffraction spot contains information derived from all the atoms in the structure; and any atom contributes to all the diffraction spots Biochemistry: Protein Methods II

  20. The phase problem Fhkl • Note that we saidIhkl = C*|Fhkl|2 • That means we can figure out|Fhkl| = (1/C)√Ihkl • We can’t figure out the direction of F:Fhkl = ahkl + ibhkl = |Fhkl|exp(ihkl) • This direction angle is called a phase angle • Because we can’t get it from Ihkl, we have a problem: it’s the phase problem! bhkl  ahkl Biochemistry: Protein Methods II

  21. What can we learn? • Electron density map + sequence  we can determine the positions of all the non-H atoms in the protein—maybe! • Best resolution possible: Dmin =  / 2 • Often the crystal doesn’t diffract that well, so Dmin is larger—1.5Å, 2.5Å, worse • Dmin ~ 2.5Å tells us where backbone and most side-chain atoms are • Dmin ~ 1.2Å: all protein non-H atoms, most solvent, some disordered atoms; some H’s Biochemistry: Protein Methods II

  22. What does this look like? • Takes some experience to interpret • Automated fitting programs work pretty well with Dmin < 2.1Å ATP binding to a protein of unknown function: S.H.Kim Biochemistry: Protein Methods II

  23. How’s the field changing? • 1990: all structures done by professionals • Now: many biochemists and molecular biologists are launching their own structure projects as part of broader functional studies • Fearless prediction: by 2020: • crystallographers will be either technicians or methods developers • Most structures will be determined by cell biologists & molecular biologists Biochemistry: Protein Methods II

  24. Macromolecular NMR • NMR is a mature field • Depends on resonant interaction between EM fields and unpaired nucleons (1H, 15N, 31S) • Raw data yield interatomic distances • Conventional spectra of proteins are too muddy to interpret • Multi-dimensional (2-4D) techniques:initial resonances coupled with additional ones Biochemistry: Protein Methods II

  25. Typical protein 2-D spectrum • Challenge: identify whichH-H distance is responsible for a particular peak • Enormous amount of hypothesis testing required Prof. Mark Searle,University of Nottingham Biochemistry: Protein Methods II

  26. Results of NMR studies • Often there’s a family of structures that satisfy the NMR data equally well • Can be portrayed as a series of threads tied down at unambiguous assignments • They portray the protein’s structure in solution • The ambiguities partly represent real molecular diversity; but they also represent atoms that area in truth well-defined, but the NMR data don’t provide the unambiguous assignment Biochemistry: Protein Methods II

  27. Comparing NMR to X-ray • NMR family of structures often reflects real conformational heterogeneity • Nonetheless, it’s hard to visualize what’s happening at the active site at any instant • Hydrogens sometimes well-located in NMR;they’re often the least defined atoms in an X-ray structure • The NMR structure is obtained in solution! • Hard to make NMR work if MW > 35 kDa Biochemistry: Protein Methods II

  28. What does it mean when NMR and X-ray structures differ? • Lattice forces may have tied down or moved surface amino acids in X-ray structure • NMR may have errors in it • X-ray may have errors in it (measurable) • X-ray structure often closer to true atomic resolution • X-ray structure has built-in reliability checks Biochemistry: Protein Methods II

  29. Cryoelectron microscopy • Like X-ray crystallography,EM damages the samples • Samples analyzed < 100Ksurvive better • 2-D arrays of molecules • Spatial averaging to improve resolution • Discerning details ~ 4Å resolution • Can be used with crystallography Biochemistry: Protein Methods II

  30. Circular dichroism • Proteins in solution can rotate polarized light • Amount of rotation varies with  • Effect depends on interaction with secondary structure elements, esp.  • Presence of characteristic  patterns in presence of other stuff enables estimate of helical content Biochemistry: Protein Methods II

  31. Poll question: discuss! Sperm whale myoglobinPDB 2jho1.4Å16.9 kDa • Which protein would yield a more interpretable CD spectrum? • (a) myoglobin • (b) Fab fragment of immunoglobulin G • (c) both would be fully interpretable • (d) CD wouldn’t tell us anything about either protein Anti-fluorescein FabPDB 1flr1.85 Å52 KDa Biochemistry: Protein Methods II

  32. Ultraviolet spectroscopy • Tyr, trp absorb and fluoresce:abs ~ 280-274 nm; f = 348 (trp), 303nm (tyr) • Reliable enough to use for estimating protein concentration via Beer’s law • UV absorption peaks for cofactors in various states are well-understood • More relevant for identification of moieties than for structure determination • Quenching of fluorescence sometimes provides structural information Biochemistry: Protein Methods II

  33. X-ray spectroscopy • All atoms absorb UV orX-rays at characteristic wavelengths • Higher Z means higher energy, lower for a particular edge Biochemistry: Protein Methods II

  34. X-ray spectroscopy II • Perturbation of absorption spectra at E = Epeak +  yields neighbor information • Changes just below the peak yield oxidation-state information • X-ray relevant for metals,Se, I Biochemistry: Protein Methods II

  35. Solution scattering • Proteins in solution scatter X-rays in characteristic, spherically-averaged ways • Low-resolution structural information available • Does not require crystals • Until ~ 2000: needed high [protein] • Thanks to BioCAT, SAXS on dilute proteins is becoming more feasible • Hypothesis-based analysis Biochemistry: Protein Methods II

  36. Fiber Diffraction • Some proteins, like many DNA molecules, possess approximate fibrous order(2-D ordering) • Produce characteristic fiber diffraction patterns • Collagen, muscle proteins, filamentous viruses Biochemistry: Protein Methods II

More Related